Field of the Invention
The present invention relates to stabilizing and protecting of biologic materials during harsh storing and use conditions, and more particularly, the invention relates to embedding bioactive materials and biologics including live bacteria in a protective formulation of an amorphous glassy matrix.
Related Art
Freeze-drying has traditionally been the most common method of preserving sensitive biological substances such as live or dead bacteria and viruses and proteins, whereas other methods such as spray drying, fluidized spray drying and desiccation are generally not suitable. The high drying temperatures used in these methods result in significant damage to the bioactive material itself. In addition, they may not sufficiently dry the material to the specific residual moisture or water activity requirements for product stability, and thus an additional drying step by other means, may be required. A conventional freeze drying process typically involves freezing the solution containing the bioactive material, and lyophilizing the frozen biomaterial under full vacuum while it remains frozen. The low temperatures of the freeze-drying process decrease the degradation reaction of the bioactive material and minimize the loss of activity in the final dry form. Often the freeze drying process results in a significant loss of activity and damage to the bioactive material due to the formation of ice crystals during the slow drying process. Furthermore, the freezing step itself, if not done correctly, can denatured or inactivate the bioactive material. Damage caused by the formation of an ice crystals structure may be circumvented, to a certain degree, by the addition of cryoprotective agents to the bioactive solution (Morgan et al., 2006). Such protective agents are highly soluble chemicals that are added to a formulation to protect cell membranes and proteins during freezing and to enhance stability during storage. Common stabilizers for live bacteria and viruses include high sugars such as sucrose, glycerol, or sorbitol, at high concentrations with the cellular material or bioactive (Morgan et al., 2006; Capela et al., 2006). However, such protective agents may not penetrate adequately into the cell to protect active components within the intracellular volume which may lead to instability upon storage of the freeze-dried substances. For this reason, membranous biomaterials such as viruses, bacteria, and cells do not survive well in freeze-drying process. Therefore, a significant challenge remains to develop an optimal drying process and formulation that minimizes drying losses while achieving adequate storage stability of the dried material.
Some of the issues associated with the freeze-drying have been resolved by using a combination of certain formulations and vacuum drying in a glassy state, particularly sugar glasses (U.S. Pat. No. 6,190,701). The dry stabilized bioactive materials are protected in a glassy matrix against hostile environments such as high temperatures and humidity. Generally, stabilization by glass formation is initiated by concentrating the sugar solution containing a bioactive molecule to form supersaturated syrup. Further water removal progressively solidifies the syrup, which eventually turns into a solid sugar glass at low residual water content. Chemical diffusion is negligible in glass and therefore chemical reactions virtually cease. Since denaturation or membrane damages are chemical changes, they cannot occur in the glass and the bioactive material is stabilized and protected. Many glasses fail to stabilize because they react with the bioactive material during storage. Obvious problems occur with reducing sugars, which may form good physical glasses but then their aldehyde groups attack amino groups on the bioactive in a typical Maillard reaction, whereas non-reactive sugars give stable products, which require no refrigeration at all.
Since sugars are inherently hygroscopic, water removal and final drying of the supersaturated syrup become extremely difficult. This drawback was first addressed by (Annear 1962) who developed a formulation containing bacteria in a solution of sugars and amino acids and a vacuum drying process that involves boiling and foam formation of the concentrated syrup. Roser et al. (U.S. Pat. No. 6,964,771) disclosed a similar concept of drying by foam formation that includes a concentration step by evaporating the bulk of the solvent followed by boiling and foaming the concentrated syrup under vacuum. To mitigate the oxidation and denaturation damage that can occur during the boiling step, Bronshtein (U.S. Pat. Nos. 5,766,520, 7,153,472) introduced an improved protective formula containing carbohydrates and surfactants. The drying of the protective solution also involved a stepwise process of concentration under a moderate vacuum before application of a strong vacuum to cause frothy boiling of the remaining water to form dry stable foam. To circumvent the boiling step, Busson and Schroeder (U.S. Pat. No. 6,534,087) have introduced a liquid state drying process of a formulation suitable for sensitive bioactive materials and using a vacuum oven under very mild vacuum pressure above 30 Torr. After achieving a certain level of drying without boiling the material, heat was applied at above 20° C. and dried material was harvested after only a few hours.
This type of drying process, in which the bioactive solution is maintained in a liquid state during the entire drying process, has the advantage of faster drying due to evaporation of the liquid during boiling and the increased surface area presented by the foaming surfaces. However, boiling and foaming require input of a significant amount of heat to provide the necessary eruption of the solution. Such a drying process is not well adapted to drying of sensitive biologicals, such as viable viruses, cells or bacteria because, the applied heat accelerates enzymatic degradation (e.g., proteolysis), and chemical oxidation (e.g., oxidation and free radical attacks), which can destroy the activity or viability of the biological material.
The drying process described above is also limited in its ability to be scaled to a large industrial process. The avoidance of freezing requires the process to be conducted at lower vacuum level (>7 TORR) than in conventional freeze drying or spray freeze drying process cycles. The most significant disadvantage of the above processes is the inability to control and limit the expansion of the foam within the vessel, tray or vial. The uncontrollable eruption and often-excessive foam formation makes it practically impossible to develop an industrial scale process. The eruption and foaming nature of the boiling step results in a portion of material being splattered on the walls of the vessel and into the drying chamber. To soften the eruption during boiling, Bronshtein (U.S. Pat. Nos. 6,884,866, 6,306,345) has proposed special chambers and a controlled temperature/pressure application protocol that reduces overheating to an acceptable level. Another approach to contain the eruption and excessive foaming is described in U.S. Pat. Publication No. 2008/0229609, in which the bioactive solution is enclosed in a container or a bag covered with breathable membranes. Once again, these protocols are difficult to implement in industrial level, they require special equipment and are difficult to reliably reproduce with different formulations.
The dry foam process, as it known in the art, is not particularly well adapted to preservation of membranous biological materials, such as liposomes, viruses or viable cells and bacteria. Lipid membranes often prevent penetration of the protective agents into enclosed volumes or prevent adequate removal of water from the enclosed volume. Without adequate penetration of protective agents, enzymatic processes, such as proteolysis, and chemical processes, such as oxidation and free radical attacks, can destroy the activity or viability of the membranous biological material. Hypoosmotic fluids remaining within membrane enclosed volumes can promote instability of the biological material. Truong-le, Vu (U.S. Pat. No. 7,381,425) describes a freeze drying process suitable for membranous bioactives. Compositions of the invention include a polyol and a membranous bioactive material. The drying process starts by cooling the formulation to a temperature of about a phase transition temperature of the lipid membranes, reducing pressure on the formulation to form stable foam, freezing the foam, and then sublimating water from the frozen foam to provide a lyophilized dry foam composition. Secondary drying conditions can be employed to further dry the foam.
A need remains for a suitable protective formulation that can be dried in glassy state without boiling and excessive foaming. There is a need particularly for a cost effective formulation and scalable drying process that is also suitable for applications outside the pharmaceutical industry such as food and agriculture industries. Protective formulations and mild drying processes are required to provide adequate drying without exposure to high temperatures. A composition is needed that can protect such biologicals in storage under high temperature and humid conditions. The present invention provides a solution to all of these challenges as is described below. The dehydration process of the present invention is very gentle and does not expose the active agent to boiling or foaming and is therefore advantageous over conventional freeze drying and foam drying techniques which would subject the sample to one or both of these stresses.